Rice sucrose tranporter gene promoter

ABSTRACT

The present invention provides DNA having the promoter activity of an OsSUT1 gene, as well as a transgenic plant and others capable of expressing a desired foreign gene specifically in the vascular bundle or the phloem thereof, using the above DNA as a promoter.  
     According to the present invention, DNA having the promoter activity of the OsSUT1 gene was isolated from the genomic library of rice ( Oryza sativa  L.) The DNA has a promoter sequence, which has a phloem specificity and a growing time specificity and exists in a nucleotide sequence shown in SEQ ID NO: 1. A transgenic plant expressing a foreign gene specifically in the phloem or in the vascular bundle system tissues was produced by ligating the desired foreign gene downstream of the promoter DNA and incorporating it into the genome of a plant.

TECHNICAL FIELD

[0001] The present invention relates to a gene expression systemtargeting a plant, particularly targeting the vascular bundle systemtissues of monocotyledons, and more particularly targeting the phloem inthe vascular bundle. Specifically, the present invention relates to aDNA having a nucleotide sequence with the promoter activity of the ricesucrose transporter gene, an expression cassette, a transgenic planttransformed using these constructs.

PRIOR ART

[0002] Generally, the vascular system of plants has been well known asan organ for conducting assimilation products of photosynthesis orwater. In addition, it has recently been reported that the vascularsystem is associated with signal transduction at an individual plantlevel, morphogenesis and the like. Sucrose transporter (hereinafterreferred to as “SUT”) is known as a protein that is mainly located invascular bundle system tissues. SUT is a membrane protein whichtransports saccharose (sucrose) in higher plants, and it is consideredthat this protein plays a role in the active transport of sucrose, bycoupling with H⁺-ATPase localized in membranes, using the activetransport of proton (H⁺) as a driving force.

[0003] SUT gene has already been isolated from multiple plant speciesincluding dicotyledons. The plant from which SUT has been isolated forthe first time is spinach (Riesmeier J W, Willmitzer L, Frommer W B(1992), Isolation and characterization of a sucrose carrier cDNA fromspinach by functional expression in yeast. EMBO J 11: 4705-4713).

[0004] It has been confirmed that SUT isolated from a dicotyledon,Solanum tuberosum, is expressed specifically in the phloem in thevascular bundle (Riesmeier J W, Hirner B, Frommer W B (1993), Potatosucrose transporter expression in minor veins indicates a role in phloemloading. Plant Cell 5: 1591-1598). Subsequently, the expression of SUTspecific to the phloem in the vascular bundle has been reported forother plants including Plantago (Gahrtz M, Stolz J, Sauer N (1994), Aphloem-specific sucrose-H⁺ symporter from Plantago major L. supports themodel of apoplastic phloem loading. Plant J. 6: 697-706) and Arabidopsisthaliana (Sauer N, Stolz, J (1994) SUC1 and SUC2: two sucrosetransporters from Arabidopsis thaliana; expression and characterizationin baker's yeast and identification of the histidine-tagged protein.Plant J 6: 67-77). Moreover, the expression of the SUC specific to thephloem in vascular bundle has also been confirmed in other plants suchas Arabidopsis, tomato and garden pea.

[0005] The promoter of the above dicotyledon, Arabidopsis, has alreadybeen isolated, and the analysis of the expression system has beencarried out using a “promoter: GUS system” and a “promoter: GFPtransformant” system (Truernit E, Sauer N (1995), The promoter of theArabidopsis thaliana SUC2 sucrose-H⁺ symporter gene directs expressionof β-glucuronidase to the phloem: Evidence for phloem loading andunloading by SUC2. Planta 196: 564-570: Imalau A., Truernit E., Sauer N.(1999), Cell-to-cell and long-distance trafficking of the greenfluorescent protein in the phloem and symplastic unloading of theprotein into sink tissues. Plant Cell 11: 309-322).

[0006] In the case of monocotyledons, SUT gene was first isolated fromrice (Hirose T, Imaizumi N, Scofield G N, Furbank R T, Ohsugi R (1997),cDNA cloning and tissue specific expression of a gene for sucrosetransporter from rice (Oryza sativa L.), Plant Cell Physiol 38: 1389 to1396). Thereafter, SUT gene has also been isolated from maize and otherplants (Aoki N., Hirose T., Takahashi S., Ono K., Ishimaru K., Ohsugi R.(1999), Molecular cloning and expression analysis of a gene for asucrose transporter in maize (Zea mays L.). Plant Cell Physiol. 40:1072-1078).

[0007] Rice is the only monocotyledon in which SUT gene expression hasbeen confirmed to be localized in the phloem (Matsukura C. et al., 2000,Plant Physiol. 124: 85-94). Moreover, although it is not a reportregarding SUT, a RPP13-1 gene with the promoter thereof has beenisolated as a gene which is expressed specifically in the phloem(Ishiwatari Y, Fujiwara T, McFarland K C, Nemoto K, Hayashi H, Chino M,Lucas W J (1998), Rice phloem thioredoxin h has the capacity to mediateits own cell-to-cell transport through plasmodesmata. Planta 205:12-22).

[0008] In addition, there have been some reports regarding promotersthat control gene expression specific to the phloem (U.S. Pat. No.5,495,007, DE Pat. No. 4,306,832, International Publications WO93/04177,WO92/22582, WO91/09050 and WO00/11197), but none of these reports relateto SUT gene promoter.

SUMMARY OF THE INVENTION

[0009] In recent years, in the development of genetic transformationtechnology for plants, gene expression systems which use a promoterspecific to a tissue, an organ or a growth stage, has been required inplace of conventional systemic or constant gene expression systems. Inparticular, the establishment of an expression system of a useful genethat targets vascular bundle system tissues, acting also as an infectionroute of pathogenic fungi or viruses, can be expected to be an importantmeans for imparting disease resistance, converting the transportcapacity of vascular bundle by genetic transformation, improvingherbaceous type by genetic transformation, and others.

[0010] Most of all, the specific expression system, targeting vascularbundles, of a gene of interest such as an insecticidal protein,antiviral protein or disease resistance protein will be extremely usefulin rice that is an important crop. In addition, the specific expressionin vascular bundles of proteins participating in the sugar metabolism(sucrose synthase, sucrose phosphate synthase, etc.) or sugar transport(SUT, H⁺-ATPase, etc.) system or a gene encoding a sugar signaltransduction factor, which is directed towards the improvement of sugartransport through vascular bundles, and further, the specific expressionof a certain protein to promote transportation of substances through thephloem, are also desired.

[0011] Thus, it is industrially very important to construct a geneexpression system which is capable of expressing a desired gene in amanner targeting at vascular bundle system tissues of a higher plant,rice, by exploiting the controlling function of a promoter.

[0012] However, no SUT gene promoter of monocotyledons has previouslybeen isolated, and there are only a few reports of promoters whichcontrol gene expression specific to the phloem in the vascular bundle ofmonocotyledons.

[0013] It is an object of the present invention to isolate a promoterfrom major monocotyledon crops, particularly from the rice plant, whichis specific for the vascular bundle tissues, and more particularly forthe phloem thereof. It is another object of the present invention toprovide a transgenic plant with the above described various improvementsby applying the controlling function of SUT gene promoter. The presentinvention further provides a method of producing the above transgenicplant, and an expression vector as well as an expression cassette, whichcan be used in the method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows the construction of a GUS expression vectorcomprising OsSUT1 promoter.

[0015]FIG. 2 shows the running state of vascular bundles on the portionfrom the leaf blade to the leaf sheath of a rice plant. FIG. 2aschematically shows the running state of vascular bundles on a riceleaf, FIG. 2b is magnified picture of tissues corresponding to a portionof the rice leaf of FIG. 2a, and FIG. 2c is a magnified picture of atransverse vascular bundle of a leaf in a cross sectional view.

[0016]FIG. 3 is a photograph showing the activity of OsSUT1-promoter-1by GUS staining. FIG. 3a is a magnified picture of the surface of aleaf, FIG. 3b is a magnified picture of a root, FIG. 3c is a set ofmagnified pictures showing a leaf in cross sectional view, and FIG. 3dis a set of magnified pictures showing a root in cross sectional view.In the figures, L, M, T and cc represent a large vascular bundle(longitudinal), a small vascular bundle (longitudinal), a transversevascular bundle and a phloem companion cell, respectively.

[0017]FIG. 4 is a photograph showing the activity of OsSUT1-promoter-2by GUS staining. FIG. 4a is a magnified pictures of the surface of aleaf, FIG. 4b is a magnified pictures of a root, FIG. 4c is a set ofmagnified pictures showing a leaf in cross sectional view, and FIG. 4dis a set of magnified pictures showing a root in cross sectional view.In the figures, L, M and T represent a large vascular bundle(longitudinal), a small vascular bundle (longitudinal) and a transversevascular bundle, respectively.

[0018]FIG. 5 is a photograph showing the activity of OsSUT1-promoter-3by GUS staining. FIG. 5a is a set of magnified pictures showing thesurface of a leaf. The upper panel shows an individual plant in whichGUS is expressed but the expression is not specific to vascular bundles,and the lower panel shows an individual in which GUS is not expressed.FIG. 5b is a cross sectional magnified picture of the leaf, of the upperpanel of FIG. 5a. FIG. 5c is a set of magnified pictures of a root incross sectional view. In the figures, L, M and T represent a largevascular bundle (longitudinal), a small vascular bundle (longitudinal)and a transverse vascular bundle, respectively.

[0019]FIG. 6 shows the activity of OsSUT1-promoter-1 in the nodes andthe internodes during the heading period by GUS staining. FIG. 6a is across sectional photographic view of a node during the heading period.Each of FIGS. 6b and 6 c is a magnified picture of a portion of the nodeof FIG. 6a. FIG. 6d is a set of magnified picture of the surface of aninternode during the heading period, in which the left panel shows adifferentiation zone, the center panel shows an elongation zone, and theright panel shows a maturation zone. FIG. 6e schematically shows thepositions of the differentiation zone, the elongation zone and thematuration zone in an internode. In the figure, VB is a vascular bundle,P is phloem, and X is xylem.

[0020]FIG. 7 shows the activity of OsSUT1-promoter-1 in a flower organduring the flowering period by GUS staining. Each of FIGS. 7a and 7 b isa magnified picture of a flower organ region comprising a glumous flowerbefore flowering. Each of FIGS. 7c and 7 d is a magnified picture of aflower organ region comprising a glumous flower immediately afterflowering. FIG. 7e schematically shows the structure of a flower organ.In the figures, L represents a lodicule and P represents a pistil.

[0021]FIG. 8 is a photograph showing the activity of OsSUT1-promoter-1in flower organ region during the flowering and ripening periods. FIG.8a shows a stigma before flowering, FIG. 8b shows an ovary at 3 daysafter flowering, and FIG. 8c shows a pericarp vascular bundle at 10 daysafter flowering. FIG. 8d shows a longitudinal segment of a stigma beforeflowering, FIG. 8e shows a transverse segment of an ovary wall at 3 daysafter flowering, and FIG. 8f shows a transverse segment of the phloem ina pericarp vascular bundle at 10 days after flowering.

[0022]FIG. 9 shows the position of OsSUT1-promoter-1 which is underlinedin the nucleotide sequence corresponding to SEQ ID NO: 1.

[0023]FIG. 10 shows the position of OsSUT1-promoter-2 which isunderlined in the nucleotide sequence corresponding to SEQ ID NO: 1.

[0024]FIG. 11 shows the position of OsSUT1-promoter-3 which isunderlined in the nucleotide sequence corresponding to SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE INVENTION

[0025] As a result of intensive studies, the present inventors haveidentified DNA having the promoter activity of OsSUT1 gene from thegenomic library of a rice plant (Oryza sativa L.; variety: Aoinokaze)for the first time, and have determined its nucleotide sequence. Theyhave found that this promoter is capable of expressing a structural geneligated downstream thereof specifically in the phloem in the vascularbundle of a plant or in the vascular bundle during the flowering period.They produced an expression cassette comprising a foreign structuralgene ligated in the downstream of the promoter, and also produced avector comprising the expression cassette. They then succeeded inactually producing a transgenic plant, which was transformed using thevector, thereby accomplishing the present invention.

[0026] Moreover, the present inventors also have found that when aportion of the 5′-side of the above nucleotide sequence is deleted, thephloem specificity is lost and the gene is expressed in the entirevascular bundle system tissues, and that the expression specificity inrespect of growth stage is also altered. These findings mean that theOsSUT1 gene promoter exhibits different features between the 5′-side andthe 3′-side regions, suggesting that by use of the different features,an expression system which enables controlling of an expression site anda growth stage can be constructed.

[0027] Promoter

[0028] That is to say, the present invention provides a DNA whichcontains a promoter sequence existing in the nucleotide sequence of SEQID NO: 1. The term “promoter” is generally used in the presentspecification to mean a nucleotide sequence existing upstream of thetranscription initiation point of a structural gene and controlling theexpression of the gene. Thus, the term promoter also includes e.g.,transcription controlling sequences such as an enhancer.

[0029] OsSUT1-promoter-1

[0030] More specifically, the first promoter existing in the nucleotidesequence shown in SEQ ID NO: 1, named “OsSUT1-promoter-1”, is a DNA,which comprises a nucleotide sequence ranging from a nucleotide in theregion from nucleotide “285 (T) through nucleotide 296 (G)” to anucleotide in the region from nucleotide “1088 (G) through nucleotide1403 (C)” in the nucleotide sequence shown in SEQ ID NO: 1, or anucleotide sequence where a portion of the region from the 5′-endthrough nucleotide 947 (G) is deleted in the above nucleotide sequence;and has a strong site-specific promoter activity directed to the phloemsite in the vascular bundle of plants. In addition to the promoteractivity specific to the phloem in the vascular bundle in green leavesand roots, this promoter also has an activity specific to the phloem inthe vascular bundle and to growth stage, in nodes, internodes and flowerorgans during the ear emergence and flowering periods.

[0031] The present invention further provides a DNA which hybridizeswith the OsSUT1-promoter-1 under highly stringent conditions and has asubstantially identical promoter activity, especially the promoteractivity specific to the phloem in the vascular bundle tissues.

[0032] The present invention further provides a DNA, which comprises anucleotide sequence having insertion, addition, deletion or substitutionof one or more nucleotides in the nucleotide sequence of theOsSUT1-promoter-1, and has a substantially identical promoter activity,especially the promoter activity specific to the phloem in the vascularbundle tissues.

[0033] The present invention further provides a DNA, which is highlyhomologous with the nucleotide sequence of the OsSUT1-promoter-1, andhas a promoter activity substantially identical to theOsSUT1-promoter-1, especially the promoter activity specific to thephloem in the vascular bundle tissues. The nucleotide sequence of theDNA shows at least 80%, preferably 85% or more, and more preferably 95%or more homology with that of the OsSUT1-promoter-1. The level ofhomology (%) can be determined using, for example, the BLAST programdescribed in Altschul et al., Nucl. Acids. Res. 25., pp. 3389-3402(1997). The BLAST program can easily be obtained through the internet.

[0034] OsSUT1-Promoter-2

[0035] The second promoter existing in the nucleotide sequence shown inSEQ ID NO: 1, named “OsSUT1-promoter-2”, is a DNA, which comprises anucleotide sequence from “T at nucleotide 948 through C at nucleotide1403” in the nucleotide sequence shown in SEQ ID NO: 1 and has apromoter activity specific to the vascular bundle tissues of plants.This promoter has a low phloem-specificity, but maintains thespecificity to the vascular bundle tissues of plants. For example, thispromoter has activity specific to the phloem, xylem and parenchymatouscells in the vascular bundles of green leaves and roots. Moreover, thevascular bundle system tissue-specific activity of this promoter tendsto less dependent on growing period and organ when compared with that ofthe above first promoter.

[0036] The present invention further provides a DNA which hybridizeswith the OsSUT1-promoter-2 under highly stringent conditions and has asubstantially identical promoter activity, especially the promoteractivity specific to the plant vascular bundle tissues.

[0037] The present invention further provides a DNA, which comprises anucleotide sequence comprising insertion, addition, deletion orsubstitution of one or more nucleotides in the nucleotide sequence ofthe OsSUT1-promoter-2, and has a substantially identical promoteractivity, especially the promoter activity specific to the plantvascular bundle tissues.

[0038] The present invention further provides a DNA, which is highlyhomologous with the nucleotide sequence of the OsSUT1-promoter-2, andhas a promoter activity substantially identical to theOsSUT1-promoter-2, especially the promoter activity specific to theplant vascular bundle tissues. The nucleotide sequence of the DNA showsat least 80%, preferably 85% or more, and more preferably 95% or morehomology with that of the OsSUT1-promoter-2. The level of homology (%)can be determined using, for example, the BLAST program described inAltschul et al., Nucl. Acids. Res. 25., pp. 3389 to 3402 (1997). TheBLAST program can easily be obtained through the internet.

[0039] It should be noted that TATTATA from nucleotides 1119 to 1125 ofSEQ ID NO: 1 is assumed to be TATA box. Accordingly, in a preferredembodiment of the present invention, OsSUT1-promoter-1 andOsSUT1-promoter-2 extend such that the 3′-end reches A at nucleotide1125 or a nearby nucleotide thereto in SEQ ID NO: 1.

[0040] Preparation of Promoter

[0041] Isolation, purification and cloning of OsSUT1 promoter, theconstruction of the expression vector, the transformation andregeneration of the plant cells, and others of the present invention canbe carried out by those skilled in the art by referring to the examplesdisclosed in the specification, or by techniques known in the field ofgenetic engineering. Those skilled in the art can easily understanddetailed experimental methods, when they refer to documents cited in thepresent specification or other appropriate documents.

[0042] For example, the DNA having the OsSUT1 promoter activity of thepresent invention can be obtained as follows. Using, as a probe, theentire or a part of appropriate promoter activity portion of thenucleotide sequence shown in SEQ ID NO: 1, a genomic DNA library of amonocotyledon, preferably a rice plant (Oryza sativa L.), may bescreened by a method such as colony hybridization. A DNA of interest maybe selected from among colonies comprising a DNA fragment, e.g. with alength of approximately 1,000 bp, and hybridizing with the probe.

[0043] Conditions for the hybridization should be “highly stringent.”That is to say, hybridization conditions with a hybridization buffer(0.5 M Na₂HPO₄, 7% SDS, 1% polyvinylpyrrolidone) and a temperature ofapproximately 63° C. or higher, preferably approximately 65° C. orhigher are prefered. By repeating the screening twice or more, andpreferably three times or more under such hybridization conditions, itis possible to obtain a DNA, which has a nucleotide sequence identicalto, or highly homologous with, for example, approximately 85% or more,and preferably approximately 95% or more homologous with the nucleotidesequence of the above OsSUT1-promoter-1 or -2: and has a substantiallyidentical promoter activity.

[0044] Moreover, a DNA comprising “a nucleotide sequence comprising aninsertion, addition, deletion or substitution of one or severalnucleotides” with respect to the nucleotide sequence of the aboveOsSUT1-promoter-1 or -2, can easily be obtained from theOsSUT1-promoter-1 or -2, or from a DNA homologous therewith, by e.g.,site-directed mutagenesis. Such mutagenesis can be carried out not onlyby the above site-directed mutagenesis, but also by any method known toa person skilled in the art.

[0045] The obtained DNA fragment is amplified by a known technique, PCR,and its nucleotide sequence is determined. Subsequently, by comparingthe determined sequence with the nucleotide sequence of SEQ ID NO: 1, itcan be confirmed that it is a promoter of interest.

[0046] In order to directly confirm the promoter activity of the DNAfragment, a reporter gene is ligated downstream of the 3′-side of theDNA so as to produce an expression vector, and the obtained expressionvector is then introduced into a plant cell. Examples of reporter genes,the expression of which in a transgenic plant is detected, include GUS,LacZ and Cat genes, as their expression can be easily assayed andquantitated; and reporter genes also include those which can be testedof the tissue specificity in the expression such as above GUS gene, GFPgene and other genes. An established known assay can be used for eachreporter gene. When the expression specificity of the DNA fragment isconfirmed by examining the promoter activity, the sequence of the DNAfragment can be selected as a “nucleotide sequence of the expectedregion having a promoter activity” for the following selection ofdesired expression specificity.

[0047] Expression Cassette and Vector

[0048] The DNA having the promoter activity according to the presentinvention can be used as an expression cassette together with a foreigngene, which is to be expressed in a manner specific to phloem in avascular bundle, the vascular bundle, or an organ, or a growth stage.

[0049] Any gene other than OsSUT1 gene can be used as a “foreign gene”,as long as it can be expressed in the above vascular bundle, especiallyin the phloem in the vascular bundle under the control of the DNA havingthe promoter activity of the present invention. A typical exampleincludes a gene encoding a protein, which has a useful action when thegene is expressed specifically in the area of the vascular bundle.Focusing attention on the fact that a vascular bundle acts as aninfection route, examples of a preferred foreign gene include proteinswhich impart disease resistance to plant bodies. Among them, ananti-pest protein, antibacterial protein or antiviral protein isespecially useful. Paying attention to the fact that the formation of avascular bundle system plays an important role in the morphogenesis ofan individual plant, it is also possible to express a gene controllingherbaceous height or herbaceous type, targeting the entire vascularbundle. Examples of particularly preferred genes include GAI geneassociated with the signal transduction of gibberellin, and the like.

[0050] A method of constructing an expression cassette by ligating aforeign gene of interest to the 3′-side of a promoter region is known toa person skilled in the art. The constructed cassette is then amplifiedin an appropriate vector (e.g., pBSII, or other vectors belonging to thesame type). The amplified expression cassette can be incorporated in thegenome of a plant cell by homologous recombination, for example, by wayof an intermediate vector for recombination. The constructed cassette isintroduced into Escherichia coli or the like for amplification.Moreover, the cassette can be introduced into Agrobacterium by triplecross, using Agrobacterium and two types of Escherichia coli (one ofwhich is Escherichia coli containing the cassette).

[0051] The above described expression cassette may comprise, asappropriate, an enhancer sequence, untranslated regions at the 5′- and3′-sides of a foreign gene, and others. Moreover, the expressioncassette may further comprise a marker gene to select a transformant.Examples of marker genes include resistance genes for antibiotics suchas tetracycline, ampicillin, kanamycin, neomycin, hygromycin orspectinomycin; luciferase gene; β-galactosidase gene; β-glucuronidase(GUS) gene; green fluorescence protein (GFP) gene; β-lactamase gene;chloramphenicol acetyltransferase (CAT) gene; and others.

[0052] Introduction of Expression Cassette into Plant Cell

[0053] The present invention also provides a transgenic plant in whichthe above described expression cassette is incorporated into the genomeof said plant transformed with the above described expression vector,whereby the promoter activity allows a foreign gene to be expressedspecifically in the vascular bundle tissues, especially in the phloemthereof.

[0054] A method of introducing a foreign gene into a plant cell genomeusing the above described expression cassette is not particularlylimited, and examples of the method include Agrobacterium method,electroporation, PEG method, microinjection, particle gun method andothers. A preferred method is the Agrobacterium method. The details ofthe Agrobacterium method are described in e.g., PCT InternationalPublication WO92/13957. As will be described in examples later, thismethod involves the application of the native transformation ability ofAgrobacterium. The method preferably comprises transferring anexpression cassette into Agrobacterium by conjugation or the like froman intermediate vector containing the expression cassette, and theninfecting a plant with this Agrobacterium. The method of infecting aplant with Agrobacterium is well known to a person skilled in the art,and examples of the method include a method of partly damaging planttissue and infecting the plant with bacteria therefrom, a method ofinfecting the embryonic tissues (including immature germ cells) of aplant with bacteria, a method of infecting a plant callus with bacteria,a method of coculturing protoplasts and bacteria, and a method ofculturing a segment of leaf tissues with bacteria (leaf disk method).

[0055] The obtained transformed cell can be selected from among othercells by using an appropriate marker as an index, or by determiningwhether or not it expresses a desired character. A method of culturingthe transformed plant cell in a regeneration medium to obtain a completeplant is described in e.g., Y. Hiei et al., Plant J. 6. 271-282: 1994.

[0056] The analysis of the obtained transformed plant can be carried outby various methods well known to a person skilled in the art. Forexample, oligonucleotide primers are synthesized on the basis of the DNAsequence of the introduced gene, and then PCR is carried out using theseprimers to analyze the chromosomal DNA of the transgenic plant.Moreover, the analysis can also be carried out by checking the presenceor absence of mRNA or protein expression corresponding to the introducedgene. Furthermore, the analysis can also be carried out from theappearance of the plant (for example, where a gene encoding a proteincapable of generating local necrotic spots is introduced, the presenceor absence of the local necrotic spots, or the size or number of thespots, etc., can be checked), disease resistance (for example, thepresence or absence of resistance or the resistance level when the plantis contacted with pathogenic fungi), and others.

[0057] By way of an example, a foreign gene can be introduced into thegenome of a plant cell together with the promoter of the presentinvention by the following steps:

[0058] (a) providing a promoter of the present invention;

[0059] (b) ligating a desired structural gene downstream of the abovepromoter, and, if necessary, further ligating a selection marker geneafter the above structural gene so as to produce an expression cassette;

[0060] (c) introducing the above expression cassette into the genome ofa plant by the Agrobacterium method, electroporation, PEG method,microinjection or particle gun method;

[0061] (d) screening transformed plant cells by the selection marker andculturing the obtained cells to form a callus;

[0062] (e) culturing the generated callus in a regeneration medium untilit becomes a complete plant; and

[0063] (d) as desired, breeding the obtained plant by self-pollinationor cross-pollination, and thereby establishing a variety of homozygoustransgenic plants.

[0064] It should be noted that the term “transformant” is used in thepresent specification to include not only transformants which areobtained by the method of the present invention comprising obtainingrecombinant plant cells and then regenerating a plant from the plantcells, but also progeny plants obtained from the transformants, as longas their expression specificity is maintained. The term “plant” is usedherein to include, unless otherwise specified, not only a plant(individual), but also seeds (including germinated seeds and immatureseeds), organs or portions thereof (including leaf, root, stem, flower,stamen, pistil and segments thereof), plant culture cells, calluses, andprotoplasts.

ADVANTAGES OF THE INVENTION

[0065] The present invention provides a DNA having a promoter activityextremely useful for higher plants and an expression cassette containingthe DNA, and a novel gene expression system, which has not beenaccomplished with the conventional vascular bundle expression system,are thereby provided.

[0066] In particular, the use of OsSUT1-promoter-1 as a promoter enablesa gene of interest to be expressed specifically in the phloem in thevascular bundle of rice plants. Moreover, OsSUT1-promoter-1 is usefulalso as a gene expression promoter targeting the phloem companion cellsin the vascular bundle during the reproductive period (reproductiveorgans, nodes and internodes during the ear emergence and floweringperiods).

[0067] Furthermore, the use of OsSUT1-promoter-2 as a promoter enables agene of interest to be expressed specifically in the vascular bundlesystem regardless of the growth stage and the type of organs. Thisactivity is useful for allowing an expression targeting the vascularbundle at an individual plant level, for example, for an expressionsystem directed towards the improvement of the transport capacity ofvascular bundles, the modification of herbaceous type, and others.

[0068] Still further, by a combined use of these promoters, it ispossible to promote expression of a gene of interest in such a mannerthat the specificity is selected to individual fine tissues (e.g.,phloem, xylem or vascular parenchymatous cells) within the vascularbundle system or to the growth stage.

[0069] The term “vascular bundle” is used herein to include: vascularbundle sheath (including vascular parenchymatous cells) which isconsisted of phloem (including phloem companion cell or phloem liquidand acting as an organ for conducting assimilation products or water)and xylem; longitudinal and transverse vascular bundles running on greenleaves of a rice plant such as a leaf sheath or leaf blade; and anyother vascular bundles existing in organs such as glumous flower, pistil(stigma), filament, lodicule or ovary wall (pericarp) during theflowering and maturation periods (however, the gene is expressed in theentire ovary wall tissues in the initial stage of maturation and around3 days after flowering). Moreover, the term “specific to the vascularbundle” is used to mean that gene expression is almost specific to theabove described vascular bundle sheath, but a preferred promoter isspecific especially for the phloem tissues in the vascular bundle.Further, the term “specific to the growth stage”, is used to mean thatthe promoter of the invention is specific to the node and internodeduring the period of internode elongation, or specific to certainperiods such as the periods of heading or flowering/ripening. Moreover,the term “specific to the growth stage” is sometimes used to mean thatthe promoter is specific to the phloem in nodes or internodes, orspecific to certain organ(s) during heading or flowering period.Therefore, in one embodiment of the invention, the specificity ofpromoters having both site and time-specificities is also included inthe definition of the term “expression specificity” in thespecification.

[0070] The promoter of the present invention with the above-describedfeatures is useful, when an anti-pest protein, antiviral protein,disease resistance protein or the like is expressed so as to impartdisease and insect damage resistance to plants. Moreover, the promoterof the invention is also useful, when a sugar metabolism (sucrosesynthase, sucrose phosphate synthase, etc.) or a sugar transport (SUT,H⁺-ATPase, etc.) system protein or a gene encoding a sugar signaltransduction factor is expressed specifically in the vascular bundle soas to improve glucose transport environment through the vascular bundle.In addition, the present promoter is useful particularly when a proteinis expressed for the purpose of transporting substances through thephloem.

EXAMPLES

[0071] Isolation of OsSUT1 Genomic Clone

[0072] Using a genomic DNA extraction kit, ISOPLANT (Nippon Gene Co.,Ltd.), genomic DNA was isolated and purified from the green leaf of arice variety (Oryza sativa L.), “Aoinokaze”. DNA (100 μg) was partiallydigested with a restriction enzyme, Sau3AI (Pharmacia Corp.). The thusobtained DNA was concentrated by isopropanol precipitation, and thensubjected to centrifugation on the continuous density gradient of 20 to5% (w/v) NaCl to provide fractions each in a small volume. The DNA sizeof each fraction was determined by agarose gel electrophoresis.Thereafter, the DNA in fractions of a size of 23 to 15 kbp was ligatedinto the XhoI site of λ BlueSTAR vector (trade name; manufactured byNovagen), and then a genomic library was produced by package mix usingGigapack Gold (trade name; manufactured by Stratagene). Using, as aprobe, a BamHI-EcoRI fragment (814 bp) that is a portion of an OsSUT1cDNA sequence, the genomic library was screened by a conventional method(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989): Molecularcloning: A laboratory manual, second ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Three positive clones were obtained.Each of these clones was digested with randomly selected 5 types ofrestriction enzymes, and thereafter, Southern hybridization analysis wascarried out using the BamHI-EcoRI fragment (814 bp) from the OsSUT1 cDNAsequence as a probe. As a result, an EcoRI fragment (4 kbp) showing thestrongest signal was isolated as a fragment containing the OsSUT1structural region. The nucleotide sequence of the obtained fragment wasdetermined using ABI Prism™ Bigdye™ Terminator Cycle Sequencing Kit and373A DNA Sequencer (both products manufactured by Applied Biosystems(PERKIN ELMER)). As a result, it was found that the nucleotide sequencecomprised the 5′-side structural region of OsSUT1 and the 1403 bpupstream region flanking to the translation initiation site (refer toFIG. 9).

[0073] Construction of Vector to Examine OsSUT1 Promoter Activity

[0074] Synthetic primers were designed so as to comprise restrictionsite sequences at both ends. Using the synthetic primers and Takara EXTaq polymerase (trade name; manufactured by TaKaRa Shuzo Co., Ltd.), thepromoter region of OsSUT1 was amplified by PCR. The sequences of thesynthetic primers employed are as follows: [Forward primer] 1.5′-TCGGCG GCC GCG ATA TCG AAT TCG CTA GGC GT-3′ (underline: NotIrestriction site) 2. 5′-AGCTCT AGA GTT GGC AGG TGC ACC ACC CTT-3′(underline: XbaI restriction site) [Reverse primer] 3. 5′-CTGGGA TCC GCCATG GCG GAC GCG CCA CG-3′ (underline: BamHI restriction site)

[0075] When the translation initiation site was set as the origin ·+1,using the above primers 1 and 3, an amplified fragment of −1119 to +14bp (1133 bp) was obtained, and using the above primers 2 and 3, anotheramplified fragment of −315 to +14 bp (329 bp) was obtained. Moreover,the amplified fragment of 1133bp was cleaved with a restriction enzymeXbaI to obtain a fragment of −455 to +14 bp (496 bp) containing thetranslation initiation site. These three fragments were cleaved with therestriction enzymes which corresponded to the linkers as shown below.

[0076] DNA fragment 1: a 1133 bp fragment: NotI and BamHI

[0077] DNA fragment 2: a 469 bp fragment: BamHI

[0078] DNA fragment 3: a 329 bp fragment: XbaI and BamHI

[0079] As shown in FIG. 2, each of these three amplified fragments wasinserted into the restriction enzyme site of pBSII vector, and a GUS-NOSgene fragment (derived from pBI121) cleaved with SmaI and EcoRI wasfurther ligated downstream thereof. Subsequently, both ends of the threetypes of promoter sequences and the region ligated to the GUS structuralregion were sequenced using ABI Prism™ Bigdye™ Terminator CycleSequencing Kit and 373A DNA Sequencer (both products manufactured byApplied Biosystems; the present PERKIN ELMER). As a result, thesequences of the both ends matched with the above genomic sequence, andit was therefore confirmed that nucleotide sequences of interest wereamplified. Moreover, no abnormality such as a frame-shift was observedat the region linking to the GUS structural region.

[0080] Each of the above DNA fragments and the above GUS structuralregion were incorporated into a vector, and the vector was thenamplified. Thereafter, in the amplified vector, transcriptional promotercontrolling regions were defined as follows. The 1108 bp upstream regionflanking to the translation initiation site was defined asOsSUT1-promoter-1 (refer to FIG. 9) for the above DNA fragment 1 (1133bp), the 456 bp upstream region flanking to the translation initiationsite was defined as OsSUT1-promoter-2 (refer to FIG. 10) for the aboveDNA fragment 2 (469 bp), and the 306 bp upstream region flanking to thetranslation initiation site was defined as OsSUT1-promoter-3 (refer toFIG. 11) for the above DNA fragment 3 (329 bp).

[0081] A cassette comprising a promoter:GUS-NOS fragment was cleavedwith NotI and HindIII from the respective vector comprising one of theDNA fragments 1 to 3, and thereafter it was inserted into thecorresponding restriction enzyme site of an intermediate vector pSB100.The thus obtained vectors were introduced into Escherichia coli LE392,and then subjected to introduction into Agrobacterium and homologousrecombination by triple cross using Agrobacterium LBA4404/pSB4 andEscherichia coli HB101/pRK2013 (Komari, T. et al., Plant J. (1996) 10:165-174).

[0082] Production of Transformed Rice by the Agrobacterium Method

[0083] Using a Japonica type rice variety “Asanohikari”, theAgrobacterium method was carried out as previously reported (Hiei, T. etal., (1994) Plant J. 6: 271-282). The obtained transformant was grown inan air-conditioned green house (lighting period: 16 hours; temperature:28° C. (day), 23° C. (night)).

[0084] GUS Staining

[0085] After transformation, the obtained callus was cultured in an N6medium containing hygromycin (50 g/ml) for redifferentiation for 4 or 5weeks, and thereafter the selected plants were used as transformants inGUS staining. The redifferentiated young plants were grown until the 5-or 6-leaf stage, and the green leaves and roots were subjected to GUSstaining according to the method of Kosugi, S. et al. (Plant Science(1990) 70: 133-140). The preparation of the section used to analyzetissue-specific expression was carried out according to the method ofMurakami et al. (Syokubutsu Saibo Kogaku (1992) 4: 281-286), usingMicroslicer DTK-1000 (Dohan E M).

[0086] Analysis of Promoter Expression by Staining (Refer to FIGS. 2 to7)

[0087] Three groups of rice plants were selected, each of whichconsisted of thirty rice plants where OsSUT1-promoter-1, -2 or -3 wasintroduced, respectively. Then, rice plants which were positivelystained by GUS were subjected to the detailed observation of tissuespecificity. As shown in FIG. 2, in the rice vascular bundle systemtissues, large vascular bundles surrounded by small vascular bundlesbetween a leaf blade and a leaf sheath run in the longitudinaldirection, and transverse vascular bundles run so that they link tothese in the transverse direction. In each photograph in the figure,symbol VB represents a longitudinal vascular bundle, symbol L representsa large vascular bundle (longitudinal) in the longitudinal vascularbundles, symbols TV and T represent transverse vascular bundles, andsymbol M represents a small vascular bundle in the longitudinal vascularbundles.

[0088] As shown in FIG. 3, in the rice plant into whichOsSUT1-promoter-1 was introduced, strong expression was observed in thephloem in the vascular bundle of the green leaves. In the photograph,the phloem which is located in the center of both in the longitudinalvascular bundle M and in the transverse vascular bundle T, wasselectively stained and so a very contrasting pattern was formed. Thisis consistent with the result of in situ analysis (Matsukura, C. et al.,Plant Physiol. (2000) 124: 85-94) using the cDNA of OsSUT1 as a probe,and it is therefore considered that these results reflect the actualexpression of OsSUT1.

[0089] In contrast, as shown in FIG. 4, being different from the case ofOsSUT1-promoter-1, the rice plant into which OsSUT1-promoter-2 wasintroduced did not show a clear pattern of light and dark between thephloem of each vascular bundle and the xylem surrounding it, but thewhole vascular bundle sheath in general was stained. That is, it wasfound that plants showed gene expression in the vascular bundle sheath(the phloem, the xylem and the vascular bundle parenchymatous cells) ofthe green leaves and roots, but that the specificity for the phloemtended to have been lost when OsSUT1-promoter-2, having a deletion of652 bp in the 5′-side area of OsSUT1-promoter-1, was introducedthereinto.

[0090] Further, with regard to plants into which OsSUT1-promoter-3,having a deletion of 802 bp in the 5′-side area of OsSUT1-promoter-1,was introduced, the number of plants which were positive by GUS stainingwas reduced overall, and as shown in FIG. 5, even in the positiveplants, the entire leaf sheath tended to be stained, thereby showinglittle specificity to vascular bundles.

[0091] To analyze growth stage specificity, the plants into whichOsSUT1-promoter-1 was introduced were successively grown in a pot, andthe gene expression until flowering was analyzed by GUS staining. As aresult, OsSUT1-promoter-1 showed strong gene expression in the phloem inthe vascular bundle of the node and the internode during the internodeelongation period (FIG. 6), and in the vascular bundle of the glumousflower, the pistil (stigma), the filament and the lodicule during theflowering period (since these are very small structures, it wasimpossible to distinguish the phloem from the xylem by this analysis)(FIGS. 7 and 8). Moreover, the gene expression was observed also in theentire ovary wall (until 3 days after flowering, especially in the upperepidermis and the lower epidermis) and the phloem in the pericarpvascular bundle (around 10 days after flowering) in the initial stage ofthe ripening period (FIG. 8). The results showing site specificity andtime specificity as described above, is consistent with the resultsobtained by Northern blot analysis (Hirose, T. et al., (1997) Plant CellPhysiol. 38: 1389-1396).

[0092] From the above results, the inventors have reached the followingconclusion:

[0093] (1) OsSUT1-promoter-1 (−1108 to −1) has a strong site-specificpromoter activity targeting the phloem in the vascular bundle. Moreover,it has the promoter activity not only in green leaves but also in nodes,internodes and flower organs during the heading, flowering and ripeningperiods, in time specific manner in addition to being specific to thevascular bundle.

[0094] (2) OsSUT1-promoter-2 (−456 to −1) has a specific promoteractivity which promotes expression of genes in the entire vascularbundle sheath of green leaves and roots, that is, in the phloem, thexylem, and the vascular bundle parenchymatous cells. The specificity forthe vascular bundle is maintained, but the specificity for the phloem islost by the deletion of a region from −1108 to −457. Accordingly, it ispredicted that a promoter sequence relating to phloem-specificexpression exists in the region of −1108 to −457. Moreover, the tendencythat phloem specificity and time specificity are relaxed by the deletionof the region so that a gene is expressed in the entire vascular bundlesheath can effectively be used for the control of the overexpression orsuppressed expression of gene targeting the parenchymatous cells and thexylem in the vascular bundle.

[0095] (3) Since OsSUT1-promoter-3 (−306 to −1) did not show vascularbundle specific expression, it is considered that the promoter sequencerelating to the expression specificity of OsSUT1 exists in a region from−1108 to −307 bp.

[0096] Construction of Expression System using the Above Promoter

[0097] To actually express a gene encoding a useful protein, astructural gene encoding a desired protein may be incorporated insteadof the above GUS-NOS sequence. At this time also, the amplification andscreening of each DNA, the construction of an expression cassette and anexpression vector, transformation, regeneration and others can becarried out by techniques well known to a person skilled in the art, asdescribed above.

1 4 1 1440 DNA Oryza sativa L. 1 tgcgcggtaa cccaccatca acctcgccgcggctttaaat gcgccgctac agggcgcgtc 60 ccattcgcca ttcaggctgc gcaactgttgggaagggcga tcggtgcggg cctcttcgct 120 attacgccag ctggcgaaag ggggatgtgctgcaaggcga ttaagttggg taacgccagg 180 gttttcccag tcacgacgtt gtaaaacgacggccagtgag cgcgcgtaat acgactcact 240 atagggcgaa ttgggtaccg ggccccccctcgaggtcgac ggtatcgata agcttgatat 300 cgaattcgct aggcgtacac cgtgaatgatttgatgcgtt gattacgggt attcatattc 360 ctttatgaaa ggttattgtc agactttttttattccacaa gatcgatcat actacaaagt 420 tattctacaa tagtttagaa cacttatccagttgtgttag aatataataa tgatggatgg 480 atatgtatgc catattaaac aatctaaattccccacaaaa catataaaag aacactataa 540 taaactatgg tttatccaac atggacatatatttaaatga agtgcgatct ccggtgctct 600 ttactggtag gatgaatgat gatagagataaaagcgttta acaaatatgg cctcaagcga 660 aattcgttat attaattaaa tcaatgaaaacatttactgg attaataaaa ctccatgcta 720 ctccattata aatgaacgca cacctatatatagcaaaatt cctatttgcc agtaggtcca 780 atacttcgga tctgtttttt tttcttttaaatatccaaaa ttgattttgg ataactactc 840 gacagtacaa acgaattaaa ccagctattacaacgtcgag tggatttaaa acactcctct 900 attaaattca cctacagaaa gtcgttcccgctgaaataat cgcaccgtct agaagctcgg 960 caagcgtgtc gctaatccga tactaactccattaattcca ttttcatttc aataattgtt 1020 gaagttatta ctgcactgga aataataaaggcaggggggt gtaactgggt gtgtacaaag 1080 tgttggtgag catagcagtt ggcaggtgcaccacccttta ttatattcct cctttctctc 1140 tctctctctc tctctctccc cctcttcctccctttaaatg cttcgcctct ctcgctcgtc 1200 tctccaaaca caaacccacc acctcctcctcctcctccca tccagcacgc gcctcctctc 1260 tcgcgcggct ttccatttcc atctccccctcctcctccta cgtctccgcc gctcctcact 1320 tcctccactc gatttccttt cttggcctctcctcctctga cacaggggtg tgcaggtttg 1380 tgtttgtgcg tggcgcgtcc gccatggctcgcggcagcgg ggccggagga ggcggcggcg 1440 2 32 DNA Artificial SequenceSynthetic PCR forward primer 2 tcggcggccg cgatatcgaa ttcgctaggc gt 32 330 DNA Artificial Sequence Synthetic PCR primer 3 agctctagag ttggcaggtgcaccaccctt 30 4 29 DNA Artificial Sequence Synthetic PCR reverse primer4 ctgggatccg ccatggcgga cgcgccacg 29

1. A DNA comprising a promoter sequence existing in the nucleotidesequence shown in SEQ ID NO:
 1. 2. A DNA, having specific promoteractivity in the phloem in plant vascular bundle tissues comprising anucleotide sequence of SEQ ID NO: 1 ranging from a nucleotide in theregion from nucleotide 285 (T) through nucleotide 296 (G) to anucleotide in the region from nucleotide 1088 (G) through nucleotide1403 (C), or a part of said nucleotide sequence where a portion of theregion from the 5′-end through nucleotide 953 (A) is deleted; or a DNAwhich hybridizes with said DNA under highly stringent conditions and hasa specific promoter activity in the phloem in plant vascular bundletissues.
 3. A DNA comprising a nucleotide sequence having insertion,addition, deletion or substitution of one to several nucleotides in thenucleotide sequence of SEQ ID NO: 1 ranging from a nucleotide in theregion from nucleotide 285 (T) through nucleotide 296 (G) to anucleotide in the region from nucleotide 1088 (G) through nucleotide1403 (C), and having a specific promoter activity in the phloem in plantvascular bundle tissues.
 4. A DNA comprising a nucleotide sequencehaving insertion, addition, deletion or substitution of one to severalnucleotides in the nucleotide sequence of SEQ ID NO: 1 ranging from anucleotde in the region from nucleotide 285 (T) through nucleotide 296(G) to a nucleotide in the region from nucleotide 1088 (G) throughnucleotide 1403 (C), where a portion of the region from the 5′-endthrough nucleotide 953 (A) is deleted, and having a specific promoteractivity in the phloem in plant vascular bundle tissues.
 5. A DNAcomprising a nucleotide sequence of SEQ ID NO: 1 ranging from nucleotide953 (A) through nucleotide 1403 (C), and having specific promoteractivity in plant vascular bundle tissues; or a DNA hybridizing withsaid DNA under highly stringent conditions and having a specificpromoter activity in plant vascular bundle tissues.
 6. A DNA comprisinga nucleotide sequence having insertion, addition, deletion orsubstitution of one to several nucleotides in the nucleotide sequence ofSEQ ID NO: 1 ranging from nucleotide 953 (A) through nucleotide 1403(C), and having a specific promoter activity in plant vascular bundletissues.
 7. An expression cassette which comprises the DNA according toany one of claims 1 to 6 and a foreign gene ligated downstream of theDNA.
 8. The expression cassette according to claim 7 which furthercomprises a marker gene for selecting a transformant.
 9. A vector fortransformation which comprises the expression cassette according toclaim 7 or
 8. 10. A transformed plant cell, wherein the genome of thecell has a promoter DNA according to any one of claims 1 to 6 and aforeign gene connected downstream of the promoter DNA incorporatedtherein, the foreign gene being expressed specifically in the phloem invascular bundle tissues or in the vascular bundle system tissues; acallus derived from the cell; a transformed plant derived from thecallus; or a progeny thereof.
 11. The transgenic plant cell, the callusderived from the cell, the transgenic plant derived from the callus, orthe progeny thereof according to claim 10, which is a monocotyledon. 12.A method of producing a transgenic plant expressing a foreign genespecifically in the phloem in vascular bundle tissues or in the vascularbundle system tissues, which comprises the steps of: (a) providing apromoter according to any one of claims 1 to 6; (b) ligating a desiredstructural gene downstream of said promoter, and further ligating aselective marker gene after said structural gene, if necessary, so as toproduce an expression cassette; (c) introducing said expression cassetteinto the genome of a plant by Agrobacterium method, electroporation, PEGmethod, microinjection or particle gun method; (d) screening transformedplant cells by means of a selection marker and subjecting the obtainedcells to callus culture; and (e) culturing the generated callus in aregeneration medium until it becomes a complete plant.